The development of genetic vectors has heralded the fast-growing field of somatic gene transfer. Vectors based on simple retroviruses, such as the Moloney Leukemia Virus (MLV), are often selected because they efficiently integrate into the genome of the target cell. Integration is thought to be a prerequisite for long-term expression of the transduced gene. However, currently available retroviral vectors can only integrate in actively dividing cells which severely limits their use for in vivo gene transfer. Non-dividing cells are the predomninant, long-lived cell type in the body, and account for most desirable targets of gene transfer, including liver, muscle, and brain. Even protocols attempting the transduction of hematopoietic stem cells require demanding ex vivo procedures for triggering cell division in these cells prior to infection.
In the early steps of infection, retroviruses deliver their nucleoprotein core into the cytoplasm of the target cell. Here, reverse transcription of the viral genome takes place while the core matures into a preintegration complex. The complex must reach the nucleus to achieve integration of the viral DNA into the host cell chromosomes. For simple retroviruses (oncoretroviruses), this step requires the dissolution of the nuclear membrane at mitotic prophase, most likely because the bulky size of the preintegration complex prevents its passive diffusion through the nuclear pores.
While retroviral vectors are useful for many kinds of in vitro gene transfer studies, problems including low titers limits their use for some in vitro and most in vivo studies. Further, another problem is that integration of retroviral vectors into the host genome was thought to be restricted to cells undergoing DNA replication. Thus, although retroviral vectors capable of infecting a broad class of cell types are known, cell division is necessary for the proviral integration of these vectors. This effectively restricts the efficient use of retrovirus vectors to replicating cells. Thus, retroviruses have not been utilized to introduce genes into non-dividing or post-mitotic cells.
A possible way around this obstacle was suggested by recent studies of the pathogenesis of lentiviral diseases. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. A typical lentivirus is the Human Immunodeficiency Virus (HIV), the etiologic agent of AIDS. In vivo, HIV can infect macrophages which are terminally differentiated cells that rarely divide. In vitro, HIV can infect primary cultures of monocyte-derived macrophages (MDM), and HeLa-Cd4 or T lymphoid cells arrested in the cell cycle by treatment with aphidicolin or .gamma. irradiation. Infection of these cells is dependent on the active nuclear import of HIV preintegration complexes through the nuclear pores of the target cells. This occurs by the interaction of multiple, partly redundant, molecular determinants in the complex with the nuclear import machinery of the target cell. Identified determinants include a functional nuclear localization signal (NLS) in the gag MA protein, the karyophilic virion-associated protein vpr, and a C-terminal phosphotyrosine residue in a subset of the gag MA protein.